抗人Endoglin单链抗体的制备及其在荷人卵巢癌裸鼠体内放射免疫显像初探

发布时间：2018-04-19 15:32

本文选题：Endoglin + CD105　；参考：《第三军医大学》2008年博士论文

【摘要】： 研究背景:Endoglin又名CD105,是一种细胞膜糖蛋白,是内皮细胞增殖的标志物之一。它在增殖的内皮细胞和肿瘤组织的新生血管内皮细胞中呈过表达,而在正常成熟的血管内皮细胞中不表达或弱表达。Endoglin还是转化生长因子-β(TGF-β)受体复合物的成分之一,具有调节内皮细胞对TGF的反应、促内皮细胞增殖和促血管形成等功能,与肿瘤血管生成有关。依据肿瘤的生长和转移依赖于肿瘤新生血管形成的观点,大量研究表明,通过靶向肿瘤新生血管内皮表达的Endoglin,在动物实验中实现了对移植瘤的放射免疫显像和治疗作用,证实了Endoglin可以作为体内肿瘤显像诊断和治疗的理想靶标。提高早期卵巢癌检出率是降低卵巢癌患者高死亡率的关键,但目前尚无理想的筛查或/和早期诊断卵巢癌的方法,因此建立特异的早期卵巢癌诊断方法显得尤为迫切和重要。卵巢癌是一高度血管化的恶性肿瘤,所以抗人Endoglin抗体有可能在其诊断及治疗中发挥重要作用。抗Endoglin抗体的研究中,以单克隆抗体的报道最多。但是单克隆抗体由于自身的一些缺点使得它的临床应用效果不佳。如单克隆抗体的分子量大,穿透力较弱,因有Fc段,在体内容易与正常组织的Fc受体发生非特异性结合,多为鼠源性,易致机体产生人抗鼠抗体反应,制备过程烦琐等。比较而言,单链抗体分子小、穿透力强、体内清除快、缺乏Fc段、异源性低、易于在大肠杆菌中重组表达获得足够量,易于用基因工程方法制备和修饰,通过构建双价或双特异性单链抗体来增加抗体的稳定性和亲和力,单链抗体是将药物、毒素和放射性物质导向肿瘤的很有价值的小分子抗体,因而在肿瘤诊断和治疗方面的应用越来越广泛。噬菌体表面呈现技术,是利用噬菌体表达外源基因的一项技术。它以改建的噬菌体为载体,把目的基因片段定向插入噬菌体外壳蛋白质基因区,使外源多肽或抗体表达并展示于噬菌体表面,进而通过亲和富集筛选表达有特异肽或抗体的噬菌体,然后进行DNA序列测定。它将基因型和表型、分子结合活性与噬菌体的可扩增性结合在一起,是一种高效的筛选系统。在抗原表位的模拟、蛋白质工程的改造、单链抗体的制备、药物筛选及疫苗研制等方面,具有广泛的用途。基于以上分析,本研究拟应用噬菌体表面呈现技术制备特异性抗人Endoglin单链抗体,并研究其生物学活性和体内靶向肿瘤新生血管内皮的作用,为探索早期卵巢癌诊断方法的研究奠定基础。研究目的:制备抗人Endoglin单链抗体,并检测其生物学活性;初步探讨抗人Endoglin单链抗体在荷人卵巢癌裸鼠体内肿瘤放射免疫显像中的应用。研究方法及结果:研究共分四部分。第一部分:以rhEndoglin(即重组人Endoglin胞外段)蛋白免疫Balb/c小鼠,纯化免疫成功小鼠脾脏的mRNA,用RT-PCR方法扩增出抗人Endoglin抗体的可变区基因,1%琼脂糖凝胶电泳示,重链和轻链可变区基因大小分别为约340bp和约325bp,与预期相符;用重叠延伸PCR经Linker序列把重链和轻链可变区基因连接成单链抗体基因,1%琼脂糖凝胶电泳示,该基因大小约750bp,与预期相符,并用SfiⅠ和NotⅠ双酶切该基因; 第二部分:把双酶切的抗人Endoglin单链抗体基因克隆入经同样双酶切的噬菌粒载体pCANTAB5E中,转化大肠杆菌E.coli TG1,再经辅助噬菌体M13KO7感染,成功构建抗人Endoglin单链抗体噬菌体呈现文库,库容量约为3.98×106;用rhEndoglin抗原对文库进行5轮吸附-洗脱-富集panning淘筛,继之用噬菌体ELISA筛选鉴定具有抗原结合能力的阳性重组噬菌体克隆,结果一次性从随机挑选的94个重组噬菌体克隆中筛选到37个阳性克隆,阳性克隆检出率约为39.36%;据噬菌体ELISA结果,对抗原亲和力最强的重组噬菌体克隆测序,获得单链抗体基因序列;经比对分析发现,该基因全长为738bp,共编码246个氨基酸残基。其中重链可变区基因编码122个氨基酸残基,位于单链抗体的N端,轻链可变区基因编码109个氨基酸残基,位于单链抗体的C端。两个可变区之间通过(Gly4Ser)3组成的15肽连接。经数据库查询,未发现任何与该单链抗体基因相同的序列;用ANTHEPROT V5.0软件对单链抗体蛋白的氨基酸序列进行分析,计算出目的蛋白的分子量为25925.818 Da。在重链和轻链可变区各有2个半胱氨酸,可形成2个二硫键。预测其等电点pI=9.015。并在生物医学网站,自动建模获得该基因编码蛋白的二级结构和三级结构的预测模型; 第三部分:把第二部分测序正确的重组噬菌体感染大肠杆菌E.coli HB2151,经IPTG诱导,成功表达了可溶性单链抗体蛋白,用SDS-PAGE和Western Blot鉴定分析,蛋白分子量约为29×103,集中在大肠杆菌的周质和培养上清中,且周质中蛋白表达量最高。确定最佳培养条件为:温度30℃,振摇速度250rpm,IPTG浓度1mmol/L,诱导培养5h;用HiTrap Anti-E Tag Column亲和层析纯化经IPTG诱导培养5h的细菌周质提取物中的单链抗体,用HiTrap Desalting Column更换单链抗体的缓冲液为中性磷酸盐缓冲液,SDS-PAGE鉴定纯化抗体的纯度大于95%;用PEG 8000浓缩纯化的单链抗体,之后Bradford法测抗体浓度为1.12mg/ml;用竞争性ELISA检测单链抗体相对于抗rhEndoglin单抗的抗原结合活活性,结果表明,该单链抗体能与抗rhEndoglin单抗竞争性结合rhEndoglin抗原表位,且竞争作用随单链抗体浓度增加而增强;用间接ELISA测定单链抗体的功能性亲和常数为(2.14±0.84)×107M-1;间接免疫荧光实验表明,该单链抗体能检测到HUVEC胞膜上表达的Endoglin,激光共聚焦显微镜下表现为胞膜上散在分布的绿色荧光点; 第四部分:用瘤细胞悬液皮下注射法接种SKOV3人卵巢癌细胞于裸鼠皮下,经病理组织学证实,成功建立了荷人卵巢癌裸鼠动物模型;石蜡切片的免疫组化结果发现,单链抗体能使移植卵巢癌的血管内皮胞膜呈阳性着色,表明该单链抗体与移植卵巢癌中鼠源性血管内皮有交叉反应;用预亚锡直接标记法完成单链抗体的99mTc标记,用纸层析法测定标记率为83%;从鼠尾静脉将99mTc标单链抗体注入荷瘤鼠体内,Spect仪器观察,对照组的肿瘤部位始终未显像,而实验组中,在注药后1h即可见肿瘤部位显像,注药后3h肿瘤显像最清晰;γ放射免疫计数器检测各组织的放射性计数,计算肿瘤组织放射性计数/非肿瘤组织放射性计数的比值,该比值反应了抗人Endoglin单链抗体在荷瘤鼠体内的分布特点,最高为2.96±1.71。结论:利用噬菌体表面呈现技术成功制备了抗人Endoglin单链抗体;体外实验证实该单链抗体具有较强的抗原结合能力;体内实验证实,通过该单链抗体靶向肿瘤新生血管内皮的作用,实现了荷瘤鼠体内移植卵巢癌的放射免疫显像。所以我们认为,该单链抗体有望作为靶向卵巢癌新生血管内皮的导向载体,为卵巢癌早期诊断方法的研究奠定了基础。
[Abstract]:Endoglin is also a marker of transforming growth factor - 尾 ( TGF - 尾 ) receptor complex .

Increasing the rate of early ovarian cancer is the key to reduce the high mortality rate in ovarian cancer patients , but there is no ideal method for screening or / or early diagnosis of ovarian cancer . Therefore , it is particularly urgent and important to establish a specific early ovarian cancer diagnosis method . Ovarian cancer is a highly vascular malignancy , so it is possible to play an important role in the diagnosis and treatment of ovarian cancer .

The monoclonal antibody has the advantages of small molecular weight , strong penetrating power , no specific binding to Fc receptor of normal tissue , and complicated preparation process .

The invention relates to a phage surface rendering technology , which is a technique for expressing foreign genes by utilizing a phage . The phage display screen is used as a carrier , the target gene fragment is directionally inserted into a phage coat protein gene region , and the exogenous polypeptide or antibody is expressed and displayed on a phage surface , and then a DNA sequence determination is carried out .

Based on the above analysis , the specific anti - human Endoglin single chain antibody is prepared by the phage surface rendering technique , and the biological activity and the role of the in vivo targeting tumor neovascular endothelium are studied , which lays a foundation for the exploration of early ovarian cancer diagnosis methods .

Objective : To prepare the anti - human Endoglin single chain antibody and to detect the biological activity of human ovarian carcinoma nude mice .

In the first part , Balb / c mice were immunized with rhEndoglin ( i.e . recombinant human Endoglin extracellular domain ) protein . The variable region gene of anti - human Endoglin antibody was purified by RT - PCR .

In the second part , the human Endoglin single - chain antibody gene was cloned into the phage vector pCAND5E , which was digested by the same double enzyme , transformed into E . coli TG1 , then infected with the helper phage M13KO7 .

coli HB2151 was infected with E . coli HB2151 . After IPTG induction , the soluble single chain antibody was successfully expressed . The molecular weight of purified antibody was determined by SDS - PAGE and Western Blot . The purity of the purified antibody was higher than 95 % .

In the fourth part , the nude mouse model of human ovarian cancer was successfully established by subcutaneous injection with tumor cell suspension . The results showed that single chain antibody could be used to make positive staining of vascular endothelial cell membrane of ovarian cancer .

Conclusion : The anti - human Endoglin single chain antibody has been successfully prepared by using phage surface presentation technique . In vitro experiments have shown that the single chain antibody has stronger antigen binding ability . In vivo experiments have shown that the single chain antibody can be used to target tumor neovascularization . Therefore , it is believed that the single chain antibody is expected to be used as a guide vector for targeting ovarian cancer neovascular endothelium , which lays a foundation for the early diagnosis of ovarian cancer .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R737.31

【参考文献】